Bacterial divisome protein FtsA forms curved antiparallel double filaments upon binding FtsN

During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA-FtsN interaction site in FtsA’s IA-IC interdomain cleft by X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the elongasome’s actin. We propose that curved, antiparallel FtsA double filaments together with treadmilling FtsZ filaments organise septal peptidoglycan synthesis in the division plane.


Introduction
In non-spherical, walled bacteria, cell shape is determined by a load-bearing structure counteracting turgor pressure named the peptidoglycan sacculus 1 . Insertion of newly synthesised glycan strands into the sacculus is mediated by the divisome in cell division, and by the elongasome (or rod complex) in cell elongation 2 . In Escherichia coli, both of these multiprotein complexes span the entire cell envelope, and contain the cytoplasmic, This work is licensed under a CC BY 4.0 International license. membrane-binding and filament-forming actin homologues FtsA (divisome) and MreB (elongasome) 3,4 .
In E. coli and many other bacteria, FtsA is the main membrane anchor for the cell division ring, the Z-ring, which initiates and organises division 5 . ZipA can function as an alternative membrane anchor for the Z-ring but seems to have a minor role in cell division in unperturbed cells 5 . The Z-ring is mainly formed by filaments of the tubulin homologue FtsZ [6][7][8] . FtsZ treadmilling dynamics, driven by FtsZ's GTPase activity, were shown to be essential for the initial condensation of FtsZ filaments into the Z-ring, but seem to become dispensable after partial constriction of the septum [9][10][11] .
Apart from localising FtsZ to the membrane, FtsA is also involved in the recruitment of essential divisome proteins such as FtsK, FtsN and potentially FtsQ 12,13 . Together with the bitopic membrane protein FtsN, FtsA forms an interaction that is crucial for the recruitment of divisome components 14 . Most of FtsN assembles last into the divisome and activates or regulates the bipartite septal PG synthase FtsWI via FtsQLB [15][16][17][18][19] . Small amounts of FtsN are recruited early to the divisome in a FtsA-dependent manner 20 .
FtsA has a uniquely positioned IC domain among actin homologues 21 . FtsA's IC domain is important for the interaction with the cytoplasmic tail of FtsN, which comprises ~32 amino acids in E. coli [22][23][24][25][26] . Two FtsN suppressor mutations in FtsA located in its IC domain 16,27 further support the idea that FtsN binds the IC domain of FtsA. In FtsN, a conserved stretch of basic amino acids in its N-terminal, cytoplasmic tail is required for interaction with FtsA in vitro 22,24 . In a zipA null background, in which the FtsA-FtsN interaction becomes essential, mutation of the basic stretch in FtsN was however permissible, whereas a D5N mutation was not 23 . Taken together with previous yeast two-hybrid assays 28 , it was suggested that interaction with FtsN depolymerises FtsA and, thereby, allows recruitment of downstream divisome components via binding sites that were (partially) occluded in the FtsA polymer 14,23 . FRET microscopy found the hypermorphic FtsA* mutant to be less polymeric than wildtype FtsA on supported lipid bilayers 29 . Addition of cytoplasmic FtsN peptide in those experiments however induced FtsA* polymerisation to levels comparable to wildtype FtsA, challenging the previous model that FtsN depolymerises FtsA.
Despite the uniqueness of FtsA's IC domain amongst the actin-like proteins 21 , FtsA was shown to form single protofilaments that recapitulate some structural features of bona fide actin protofilaments 30 . Double filaments were shown to be the smallest functional unit of all other known actin homologues 31 and, indeed, Thermotoga maritima FtsA was also shown to form double filaments 30 . More recently, several mutations in E. coli FtsA, originally described as ZipA suppressor mutations, were shown to facilitate double filament formation of FtsA on supported lipid monolayers 32 .
Similar to FtsA, E. coli MreB, the actin-like protein of the elongasome, binds membranes directly through an amphipathic helix and forms curved antiparallel double filaments 4,33,34 . This enables a curvature-sensing mechanism that allows MreB filaments to align with the axis of highest principal curvature, the short axis of the cell in the case of rod-shaped cells, such as Bacillus subtilis 34,35 . Hence, the elongasome uses MreB double filaments acids in E. coli) (Extended Data Figure 2a) interacted with FtsA as shown previously 20,22,23 .
Using surface plasmon resonance (SPR), we showed that EcFtsN 1-32 binds EcFtsA and EcFtsA 1-405 , a C-terminal truncation of EcFtsA lacking the amphipathic membrane binding helix, with dissociation constants (K d ) of 0.8 μM and 2.0 μM, respectively (Figure 2b). The interaction between V. maritimus FtsA 1-396 and FtsN  was about three-fold weaker than the EcFtsA 1-405 -EcFtsN 1-32 interaction (Extended Data Figure 2b). Since cross-linking of FtsA to the flow cell surface in SPR might affect FtsA polymerisation, we also probed the FtsA-FtsN interaction in solution using fluorescence polarisation (FP), for which Cterminally truncated FtsA was titrated into Atto 495-labelled FtsN peptide. FP data were fitted to a two-step model with K d s of about 0.016 μM and 11 μM for the EcFtsA 1-405 -FtsN 1-32-C-Atto 495 interaction ( Figure 2c). Again, the VmFtsA 1-396 -FtsN 1-29-C-Atto 495 interaction was about four-fold weaker for the first binding event (Extended Data Figure  2c). Next, we subjected the system to analytical ultracentrifugation using a fluorescence detection system (FDS-AUC) to characterise the two binding events of the FtsA-FtsN interaction. FDS-AUC showed that both binding events are accompanied by formation of higher order FtsA-FtsN assemblies, indicating that the cytoplasmic FtsN peptide not only binds to FtsA but could also facilitate FtsA polymerisation ( The short cytoplasmic tail of FtsN harbours two sequence motifs that are conserved among E. coli and related proteobacteria with similar FtsA and FtsN sequences: a conserved 3 R/KDY 6 (E. coli amino acid positions) motif near the N-terminus 23 and two to three stretches of basic amino acids, the most prominent of which in E. coli FtsN is 16 RRKK 19 22 (Extended Data Figure 2a, g). In E. coli, a D5N mutation in the conserved N-terminal motif of FtsN was shown to impair FtsA-FtsN interactions in vivo 23 , but did not affect binding or co-localisation with FtsA-FtsZ filaments on supported lipid bilayers in vitro 24 . In contrast, and somewhat confusingly, mutation of the basic 16 RRKK 19 stretch abrogated binding in vitro 22,24 , yet was permissible in vivo under conditions for which the FtsA-FtsN interaction becomes essential 23 . We therefore tested a set of EcFtsN 1-32 truncations and mutations in our SPR and monolayer assays to determine the effect of those modifications on the FtsA-FtsN interaction and on double filament formation (Extended Data Figure 2h, i). Mutation or deletion of any one of the three stretches of basic amino acids in EcFtsN 1-32 reduced binding affinity for FtsA and, consequently, its ability to promote FtsA double filament formation. EcFtsN 1-32, D5N and EcFtsN 1-33, scrambled (Supplementary Table T5) did not show  reduced binding affinity, but were unable to induce FtsA double filament formation. Hence,  the D5N mutation in FtsN might be non-permissible in vivo under conditions for which the FtsA-FtsN interaction becomes essential because it fails to promote formation of FtsA double filaments. In other words, this result supports the idea that FtsA double filaments are important for the activation or correct regulation of cell division by FtsA and FtsN.
A comparison between the atomic model of the symmetry-expanded helix and a cryo-EM 2D class average of EcFtsA "mini-rings" assembled on lipid monolayers shows that FtsA's IA domain is facing outwards in the "mini-rings" (Extended Data Figure 3b). In contrast to a previous study 37 , ~65 % of intact "mini-rings" we observed were comprised of 13 FtsA monomers, whereas 35 % were 12-subunit "mini-rings". Presumably because of their favourable rotational symmetry, 12-subunit "mini-rings" were mostly organised into 2D arrays as reported by Krupka and co-workers.
Due to the low resolution (3.6 Å) of the VmFtsA 1-396 -VmFtsN 1-29 co-crystal structure (Supplementary Table T1), we could not unambiguously interpret the electron density map representing about seven to eight amino acids of the VmFtsN 1-29 peptide. We therefore confirmed the location of the VmFtsN 1-29 binding site in VmFtsA 1-396 by hydrogen deuterium exchange mass spectrometry (HDX-MS) (Figure 3c). At three-fold molar excess (30 μM) of VmFtsN 1-29 over VmFtsA 1-396 , only helix 2 located in the IC domain of FtsA showed significantly reduced deuterium incorporation (lower mass differential) compared to free VmFtsA 1-396 ( Figure 3c, top left). This agrees with VmFtsN 1-29 binding across the IC domain as seen in the co-crystal structure (Figure 3d). At 10-fold molar excess (100 μM) of VmFtsN 1-29 , FtsA peptides in filament interfaces were also protected, again, most likely indicating that binding of the cytoplasmic FtsN peptide to FtsA facilitates filament formation ( Figure 3c).
As proposed previously 22 , the FtsA-FtsN interaction is reminiscent of that between PilM and PilN, two proteins involved in type IV pilus formation 39 . PilM, which also has a IC subdomain, is structurally related to FtsA, and PilN, similar to FtsN, is a bitopic membrane protein with a short, cytoplasmic tail comprising about 30 amino acids 39  To try to assign the VmFtsN 1-29 peptide sequence in the VmFtsA 1-396 -VmFtsN 1-29 cocrystal structure (PDB 7Q6I) we reasoned that based on the geometry in vivo, where FtsA binds to the cell membrane through its C-terminal amphipathic helix, the N-terminal half of VmFtsN 1-29 is expected to interact with FtsA. The globular domain of FtsA was reported to be several nanometres away from the inner membrane in E. coli 40 . The C-terminal half of the VmFtsN 1-29 peptide would be confined to the proximity of the inner membrane, as it is linked to the transmembrane helix in the full-length FtsN protein (Extended Data Figure  2a). Amino acids M1-R8 of VmFtsN 1-29 provided the best fit for the density with reasonable chemistry, i.e. several hydrogen bonds to FtsA and a hydrophobic pocket accommodating the central tyrosine (Y6) in the sequence stretch (Extended Data Figure 3d, top). This model also implies a central position of aspartate D5 within the FtsA-FtsN interaction site that could point to a mechanism by which the D5N mutation impairs double filament formation (Extended Data Figure 2h; Extended Data Figure 3d, top). In that case, FtsN D5N would not be able to bridge FtsA's IA and IC domain. We also studied the VmFtsA 1-396 -VmFtsN 1-29 interaction by nuclear magnetic resonance (NMR) spectroscopy. We assigned the peptide backbone of Gly-Gly-VmFtsN 2-29 , a VmFtsN 1-29 construct in which two glycine residues replace the N-terminal methionine (Extended Data Figure 3e). Next, we analysed peak broadening (reduction in peak intensity due to the slower tumbling rate of FtsA-bound Gly-Gly-VmFtsN 2-29 peptide) upon addition of equimolar amounts of VmFtsA 1-396 (Extended Data Figure 3f). Intensity reduction was most prominent in the stretch of amino acids Y6-G10 (Extended Data Figure 3f), suggesting a slightly shifted binding motif compared to our density-based assignment (M1-R8) (Extended Data Figure 3d). We were not able to generate a good fit of these residues into the electron density map of the VmFtsA 1-396 -VmFtsN 1-29 co-crystal structure. For technical reasons, the NMR experiments were carried out at pH 6.0, whereas binding experiments were done at pH 7.7 and crystallisation was achieved at pH 8.5.

FtsA double filaments adopt negative Gaussian curvature
Based on the observation that VmFtsA 1-396 -VmFtsN 1-29 double filaments were curved in the co-crystal structure (Figure 3b), we hypothesised that the intrinsic curvature-preference observed for MreB double filaments might also be a feature of FtsA-FtsN double filaments 4,34 . The curvature preference of MreB filaments has been proposed to enable a curvature-sensing mechanism that allows them to robustly align in cells with the axis of highest principal curvature, which corresponds to the short axis of rod-shaped bacteria such as E. coli and B. subtilis 34,35 .
To investigate the curvature preference of FtsA filaments, we added EcFtsA-FtsN 1-32 to preformed liposomes made from E. coli polar lipid extract. In the absence of proteins liposomes are typically round or oval ( Figure 4a). Liposomes coated with EcFtsA alone are also round or oval ( Figure 4b). The FtsA coat shows a 20 nm repeat indicating the presence of FtsA "mini-rings" 37 (see also Extended Data Figure 3b). In contrast, liposomes coated with EcFtsA-FtsN 1-32 double filaments showed membrane indentations with negative Gaussian curvature ( Figure 4c). We encourage the reader to revisit previously published images of MreB double filaments bound to the outside of liposomes showing a remarkably similar binding mode 4,33 . To better match the geometry inside an E. coli cell, we then encapsulated EcFtsA-FtsN 1-32 double filaments in liposomes. Protein-filled liposomes were spherocylindrical (rod-shaped) or had spherocylindrical protrusions ( Figure  4d). Spherocylinders were either ~40 nm or ~70 nm in diameter. Thicker spherocylinders contained tightly packed EcFtsA-FtsN 1-32 double filaments that were aligned with the short axis in the cylindrical section (Figure 4d, e). We again suggest the reader to compare with published images of MreB filaments inside liposomes showing similar filament arrangements 34 . EcFtsA-FtsN 1-32 filaments were regularly absent in the hemispheres (poles) of thick spherocylinders (Figure 4d, e). If filaments were present in the hemispheres, they showed random angular orientations (Figure 4e, bottom). Rarely, we obtained liposomes with only a few EcFtsA-FtsN 1-32 double filaments inside ( Figure 4f). Again, filaments were distributed randomly in spherical sections of the liposomes but aligned with the short axis of the liposomes in more cylindrical sections. We performed 2D averaging and image classification focussed on the membrane attachment sites of the FtsA filaments in thin and thick spherocylinders. Class averages clearly illustrated the different organisation of FtsA filaments in thin and thick spherocylinders ( Figure 4g). In thin spherocylinders, FtsA was organised into bent single protofilaments (Figure 4g, left). In thick spherocylinders however, FtsA formed bent double filaments, as apparent by superposition of the VmFtsA 1-396 double filament crystal structure (PDB 7Q6F) onto the 2D class average (Figure 4g, right).

E. coli FtsA forms antiparallel double filaments in vivo
To validate our in vitro findings, we investigated double filament formation of FtsA in vivo using site-specific cysteine cross-linking in E. coli. We inserted a neoR marker downstream of lpxC for positive selection (Extended Data Figure 4a). For visualisation via Western blotting, we introduced a 3x HA-tag (comprising 40 amino acids including linkers) into the H7-S12 loop of To probe the lateral association further, we generated the two, additional FtsA single cysteine mutant strains ftsA 3x HA, D123C and ftsA 3x HA, Q155C , which probe the lateral FtsA i -FtsA i* and FtsA i -FtsA i*−1 interface of the FtsA double filament, respectively ( Figure  5d). These single cysteine mutants may cross-link to their symmetry mates because of the local C2 symmetry in each of the two lateral filament interfaces. However, C β -C β distances in the VmFtsA double filament structure of 3.7 Å for FtsA 3x HA, D123C and 12.6 Å for FtsA 3x HA, Q155C indicated that cross-linking with BMOE with an expected cross-linking distance of ~8 Å might be inefficient, which prompted us to try thiol-directed cross-linkers of different lengths (Figure 5f). The ftsA 3x HA, D123C mutant showed more efficient crosslinking of FtsA using dibromobimane (dBBr) than using BMOE (Figure 5e, left). In case of the ftsA 3x HA, Q155C mutant, BMOE cross-linking did not lead to efficient formation of covalent FtsA dimers, whereas treatment with the longer maleimide cross-linkers 1,4bismaleimidobutane (BMB) and bismaleimidohexane (BMH) did (Figure 5e, right). Taken together, our data strongly suggest that FtsA forms protofilaments in cells and, that these

Conclusions
We report that FtsA polymerises to form antiparallel double filaments in E. coli and find that filament formation is induced through binding to the cytoplasmic tail of FtsN in vitro.
The only other actin-like protein known to polymerise into antiparallel double filaments is MreB, the actin homologue in the elongasome, which serves as a "rudder" to guide peptidoglycan insertion in the cell wall during growth 33,34, 31 We observed that FtsA-FtsN filaments preferentially bind surfaces of negative Gaussian curvature in and on liposomes, as do MreB double filaments 4,33,34 ( Figure 4). We therefore propose that MreB and FtsA have a common curvature sensing mechanism. Finally, we devised a model for curvature-guided cell constriction by FtsA-FtsN double filaments, which align the direction of the divisome's glycan strand synthesis activity with the circumference of the cell (Figure 6a).
In our model, we define three phases in divisome assembly and maturation. In phase 1, unaligned FtsZ filaments are present at midcell, in phase 2 a fully assembled divisome aligns with the short axis of the cell, and in phase 3 the divisome is fully activated, synthesising septal PG and enabling cell constriction ( Figure 6a). We note that the temporal order of individual recruitment and activation events during divisome maturation remains largely unknown, and could be informed by in vivo single molecule imaging of FtsA. FtsZ filaments are present at midcell, presumably to determine the division plane, but are unaligned in the absence of an alignment mechanism (Figure 6a, left). After recruitment of divisome proteins, and Z-ring condensation 10,11 , which has previously been proposed to be driven by a FtsA "mini-ring" to double filament transition 32,37 , FtsN-induced FtsA double filaments align themselves and other divisome components with the short axis of the cell. This process might be aided by FtsZ treadmilling, which could provide a long-range distribution mechanism 7,8,42 (Figure 6a, middle).
We propose that curvature-sensing FtsA double filaments provide a solution to the FtsZ alignment problem of how FtsZ filaments align with the short cell axis during division 43 . Consistently, the fraction of directionally treadmilling FtsZ filaments decreases in a ΔftsA strain of B. subtilis 10 . We hypothesise that FtsA double filaments and treadmilling FtsZ filaments align and evenly distribute divisome components in the narrow division plane. Most importantly, this could restrict movement of FtsWI, which is the bipartite PG synthase of the divisome, in such a way that cell-constricting septal PG synthesis follows the cell's circumference (Figure 6a, right). FtsN might function as an activation switch of the divisome by coordinating activities of FtsA and FtsWI. Recently, a more direct interaction between FtsA and FtsW has also been proposed 44 .
We found that the short cytoplasmic tail of FtsN promotes FtsA double filament formation (Figure 2a, d and e), which adds to previous evidence reporting that FtsA can form different polymers 30,32,37 . Importantly, our structural data reveal that the FtsA double filament is compatible with FtsN binding. Double filament formation also positions FtsA's IC domain close to the inner membrane (Extended Data Figure 1c), which might facilitate binding of divisome components such as FtsQ 24 .
Taken together with recent reports establishing FtsWI 45 and RodA-PBP2 46,47 as bipartite PG synthases, our data showing the similarities between FtsA and MreB double filaments strengthen the previously proposed evolutionary relationships between the divisome and elongasome 2 (Figure 6b). In Chlamydia, one of the few bacteria that lack FtsZ, MreB has been implicated in the organisation of cell division 48 , which further suggests that divisome and elongasome share some basic functions.
Which of FtsZ's many functions makes it the early organiser of the divisome, but are not required in the elongasome? Our model suggests that FtsZ is the long-range organiser of the division site, which ensures that septal PG synthases only function in a single division plane, and are evenly distributed around the cell's circumference. FtsA aligns the PG synthases with the orientation of the division ring, so that septal PG glycan synthesis mediated by the divisome goes around the ring's circumference.
Future studies will need to pinpoint when during cell division FtsA double filaments form.
The difference in interaction partners between monomeric and polymeric FtsA, and how they influence each other, will need to be investigated concomitantly. These studies will deepen our understanding of the central role of FtsA polymerisation in FtsZ-based cell division and bring us closer to in vitro reconstitution of bacterial cell division.

Expression plasmids
Expression plasmids (Supplementary Table T3) were cloned using NEBuilder HiFi DNA Assembly Mix (NEB). E. coli MAX Efficiency DH5α (ThermoFisher) or C41(DE3) cells (Lucigen or Sigma) were used for plasmid propagation and protein expression, respectively. Plasmid sequences are provided in Supplementary Data D1.

Protein expression and purification
The sequences of all proteins used in this study are listed in Supplementary Table T4.
Purifications were carried out at 4-6°C unless stated otherwise. Buffers were prepared in Millipore water (MPW), pH-adjusted at room temperature (RT) and filtered through a 0.22 μm PES filter.
FtsN peptides-FtsN peptides were chemically synthesised by Generon/Neobiotech. Purity (≥ 95 % in HPLC) and molecular mass were verified by the company. Lyophilised peptides were resuspended in binding buffer (50 mM HEPES/KOH, 100 mM KAc, 5 mM MgAc 2 , pH 7.7). Stock concentrations were determined using an ND-1000 spectrophotometer (NanoDrop Technologies) or a Direct Detect Infrared Spectrometer (Merck Millipore) for peptides without tyrosine and tryptophan residues. Stock concentrations were in the range of 20-70 mM. Peptide sequences are given in Supplementary Table T5.

Crystal structure determination
Crystallisation conditions were screened using our in-house high-throughput crystallisation facility 49 Supplementary Table T1. Diffraction data were collected on single crystals at Diamond Light Source (DLS), Harwell, UK, at 100 K using the in-house Generic Data Acquisition (GDA) software, as indicated in Supplementary Table T1. Diffraction data were processed using the CCP4 suite 51 Figure 3d) are provided in Supplementary Data D2.

SPR
SPR was performed using a Biacore T200 using CM5-sensor chips (Cytiva). Reference control and analyte channels were equilibrated in binding buffer. FtsA was immobilised onto the chip surface via amide coupling using the supplied kit (Cytiva) to reach a RU value of between 2000 and 7800 RU for separate experiments. Analytes were injected for 120 s followed by a 300 s dissociation in a 1:2 dilution series with initial concentrations of: 10 μM for peptide EcFtsN 1-32 ( Figure 2b); 60 μM for VmFtsN 1-29 (Extended Data Figure  2b); 20 μM for FtsN peptides in Extended Data Figure 2i. After reference and buffer signal correction, sensogram data were fitted using Prism 8.0 (GraphPad). Equilibrium response (R eq ) data were fitted to a single-site interaction model to determine K d : where C is the analyte concentration and R max is the maximum response at saturation and B is the background resonance. Fluorescence polarisation was measured using a PHERAstar FSX (BMG Labtech) directly after reaction setup and after incubation at RT for 30 min and 2 h to ensure equilibrium had been reached. Data were fitted using Prism 8.0 (GraphPad). Dissociation constants were calculated using a two-step model: where F 0 is the anisotropy in the absence of titrating protein, [P T ] is the total concentration of protein, and F Lo and F Hi are the anisotropy changes at saturation of low and high affinity sites with binding constants of K DLo and K DHi , respectively.

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Europe PMC Funders Author Manuscripts with a fluorescence optical system (Aviv Biomedical) with fixed excitation at 488 nm and fluorescence detection at > 505 nm. Data were processed and analysed using SEDFIT 16 and SEDPHAT 15.2 57 following the protocol for high-affinity interactions detected by fluorescence 58 . Binding constants were estimated using a two-step model. Data were plotted using GUSSI 1.4.2 59 .

Lipid monolayers and negative stain electron microscopy
Lipid monolayers were prepared from E. coli polar lipid extract (Avanti) 61 . Wells of a custom-made Teflon block were filled with 60 μl binding buffer. 20 μg of lipids, dissolved in chloroform, were applied on top of the buffer, and incubated for 2 min. Next, baked (60°C, overnight) CF300-Cu-UL EM grids (EMS) were placed on top of the wells with the carbon side facing downwards. Grids were incubated for 20-60 min. FtsA at 0.2 mg/ml for EcFtsA M96E, R153D and VmFtsA or at 0.1 mg/ml for all other FtsA variants was mixed with 1 mM ATP and indicated FtsN peptides at 10-fold molar excess, unless stated otherwise, in binding buffer. Samples were incubated for 10 min at RT. EM grids were carefully lifted off the buffer and blotted from the side. 4 μl sample were applied to the grid and incubated for 30 s before staining with 2% w/v uranyl formate. Grids were imaged on a Tecnai Spirit electron microscope (ThermoFisher) operating at 120 kV and equipped with a Gatan Orius SC200W camera. Presented micrographs were contrast adjusted and blurred for display purposes.

Cryo-EM of EcFtsA "mini-rings" and EcFtsA-FtsN 1-32 double filaments on lipid monolayer
Lipid monolayers were prepared as described above, with the exception that Quantifoil Au R0.6/1 300 mesh grids (Quantifoil) were used. For "mini-rings" (Extended Data Figure 3b), EcFtsA at 0.15 mg/ml was mixed with 1 mM ATP in binding buffer, and incubated for 30 min at RT. For double filaments (Figure 2f), EcFtsA at 0.1 mg/ml was mixed with 1 mM ATP and EcFtsN 1-32 at 22 μM in binding buffer, and incubated for 20 min at RT. Grids were gently blotted from the side after attachment of the monolayer and inserted into a Vitrobot Mark III (ThermoFisher) set to 20°C and 100% humidity. 3 μl of sample were applied to the grid, incubated for 30 s and blotted for 12-15 s (0.5 s drain time, -15 blot force) before plunge-freezing into liquid ethane maintained at −180°C using a cryostat 62 . For "mini-rings", grids were imaged using a Titan Krios (ThermoFisher) operating at 300 kV and equipped with a Quantum energy filter (Gatan) set to 20 eV slit width. Movies were collected on a K2-XP direct electron detector at a pixel size of 2.32 Å, −3 to −5 μm defocus and a total dose of 25 e − /Å 2 using EPU 2.13 (ThermoFisher). For double filaments, grids were imaged using a Tecnai G2 Polara (ThermoFisher) operating at 300 kV. Movies were collected on a Falcon III direct electron detector at a pixel size of 1.38 Å, −3.3 to −4 μm defocus and a total dose of 100 e − /Å 2 using EPU 1.5 (ThermoFisher).
Data were processed using MotionCor2 63 , CTFFIND4 64 and RELION-3.0 65 for double filaments or RELION-4.0 66 for "mini-rings". A total of 162,725 and 104,660 particles were automatically picked and extracted for "mini-rings" and double filaments, respectively, with the presented 2D class averages corresponding to 14,067 and 14,602 particles, respectively. Presented images were upscaled (Extended Data Figure 3b), contrast adjusted and blurred for display purposes.

HDX-MS
VmFtsA 1-396 at 10 μM was mixed with 2 mM ATP and, if indicated, VmFtsN 1-29 at 3-or 10-fold molar excess in binding buffer. 5 μL of sample were added to 40 μL of D 2 O buffer at RT for 3, 30, 300 and 1800 seconds, then quenched and frozen until further processing. Samples were rapidly thawed and subjected to pepsin cleavage followed by reversed phase HPLC separation. to 2000, with standard electrospray ionisation (ESI) source and lock mass calibrated using [Glu1]-fibrino peptide B (50 fmol/μL). The mass spectrometer was operated at a source temperature of 80°C and a spray voltage of 2.6 kV. Spectra were collected in positive ion mode. Peptides were identified by MS e67 using a 5-36% gradient of acetonitrile in 0.1% v/v formic acid over 12 min. The resulting MS data were analysed using Protein Lynx Global Server 3.0.3 (Waters) with an MS tolerance of 5 ppm. Mass analysis of the peptide centroids was performed using DynamX 3.0 (Waters). Only peptides with a score > 6.4 were considered. The first round of analysis and identification was performed automatically using DynamX 3.0; however, all peptides (deuterated and non-deuterated) were manually verified at every time point for the correct charge state, presence of overlapping peptides, and correct retention time. Deuterium incorporation was not corrected for back-exchange and represents relative, rather than absolute changes in deuterium levels. Changes in H/D amide exchange in any peptide may be due to a single or multiple amides within that peptide. Time points were prepared in parallel and data for individual time points were acquired on the mass spectrometer on the same day.
For binding studies, 1 H, 15 N BEST-TROSY spectra were acquired at 278 K on 50 μM Gly-Gly-VmFtsN 2-29 mixed with equimolar concentration of VmFtsA 1-396 in NMR buffer. As sensitivity was compromised by the formation of FtsA polymers upon binding of FtsN peptide, multiple short experiments were acquired and added up to define the ideal time window for data analysis. Each spectrum was acquired with 128 scans and a recycle delay of 400 ms, with a final spectral resolution of 4.7 Hz per points. Relative peak intensities were normalised to the C-terminal residue R29 of Gly-Gly-VmFtsN 2-29 and analysed as I bound /I free , with I bound and I free being the peak intensities of Gly-Gly-VmFtsN 2-29 with (bound) and without (free) VmFtsA 1-396 , respectively.

EcFtsA "mini-rings" and EcFtsA-FtsN 1-32 filaments on liposomes
Liposomes were prepared from E. coli polar lipid extract (Avanti) by extrusion using a Mini Extruder fitted with a polycarbonate membrane with 0.4 μm (Figure 4b) or 1 μm (Figure 4a, c) pore size (Avanti) in binding buffer (Figure 4b) or binding buffer without magnesium for EcFtsA "mini-rings" and EcFtsA-EcFtsN 1-32 filaments (Figure 4a, c), respectively. and plunge-frozen into liquid ethane maintained at −180°C using a cryostat 62 using a Vitrobot Mark III (ThermoFisher) set to 20°C and 100% humidity. Grids were imaged on a Tecnai F20 (ThermoFisher) equipped with a Falcon II direct electron detector or a Glacios (ThermoFisher) equipped with a Falcon III detector. Microscopes were operated at 200 kV and cryogenic temperature. Presented micrographs were motion-corrected (if collected on Glacios), contrast adjusted and blurred for display purposes.

EcFtsA-FtsN 1-32 filaments inside liposomes
EcFtsA-FtsN 1-32 filaments were encapsulated into liposomes by dilution of CHAPSsolubilised E. coli total lipid extract (Avanti) 40 . 50 μl of preincubated FtsA at 20 μM mixed with 200 μM FtsN 1-32 and 0.5 mM MgATP in binding buffer without magnesium were added to 50 μl of E. coli total lipid extract (Avanti) solubilised at 10 mg/ml in binding buffer without magnesium supplemented with 20 mM CHAPS. The sample was incubated at RT for 35 min, before it was gradually diluted with 500 μl of binding buffer without magnesium supplemented with 0.5 mM MgATP within 20 min. 3 μl of sample were applied to a freshly glow-discharged Quantifoil Au R2/2 200 mesh grid (Quantifoil), blotted for 5.5-7.5 s (0.5 s drain time, -15 blot force) and plunge-frozen into liquid ethane maintained at −180°C using a cryostat 62 using a Vitrobot Mark III (ThermoFisher) set to 20°C and 100% humidity. Grids were imaged on a Tecnai F20 (ThermoFisher) equipped with a Falcon II direct electron detector, operating at 200 kV and cryogenic temperature. Rarely, EcFtsA-FtsN 1-32 filaments were observed inside liposomes when added to the outside of preformed liposomes (Figure 4f), most likely due to membrane rearrangements during handling. Here, preformed liposomes extruded to 1 μm at 1 mg/ml were mixed with 0.5 mM MgATP, FtsA at 2.5 μM and FtsN 1-32 at 25 μM. Presented micrographs were contrast adjusted and blurred for display purposes. For 2D class averages (Figure 4g), movies were collected on a Glacios with a Falcon III direct electron detector at a pixel size of 1.99 Å, −2.5 to −4 μm defocus and a total dose of 56 e − /Å 2 using SerialEM 3.9 71 . Data were processed using MotionCor2 63 , CTFFIND4.1 64 and RELION-3.1 65 . Presented images were upscaled and blurred for display purposes.
Shuttle plasmid pFB483 was designed with a pMB1 origin of replication, a pheS T251A,A294G -hygR double selection cassette, a CRISPR array targeting ftsW and the region upstream of secM, and a ccdB toxin gene (outsert) flanked by BsaI acceptor sites for Golden Gate assembly 77 . CRISPR arrays were designed to mediate scarless excision. pFB483 was propagated in a ccdB survival strain.
Targeting constructs were split into 2-3 modules for insertion of single or double point mutations, respectively. Initially, the internal 3x HA-tag (120 bp including a XhoI restriction site) was inserted into ftsA using three modules, resulting in sTN001. Point mutations were introduced by PCR using sTN001 as template, and modules were assembled into pFB483 via Golden Gate assembly with BsaI. The final targeting construct also introduced a kanamycin-selectable neoR marker downstream of the lpxC gene. Assembled shuttle vectors were transformed into SFB143 and selected on LB agar plates supplemented with 200 μg/ml hygromycin-B and 50 μg/ml apramycin at 37°C.
Acceptor cells (SFB123) were grown to stationary phase in 5 ml LB supplemented with 10 μg/ml tetracycline. 4 ml of culture were harvested by centrifugation and washed three times in LB, before being transferred into 50 ml LB supplemented with 10 μg/ml tetracycline and 0.5 % w/v L-arabinose. Cells were grown at 37°C for 1h, harvested and washed three times in LB. In the meantime, donor transformants were washed off the agar plates using 2 ml LB and left at RT. All cultures were resuspended in LB to an OD 600 of 40. 12.5 μl of acceptor cells were mixed 87.5 μl of donor cells and spotted onto well-dried TYE plates. Spots were air-dried, before plates were incubated at 30°C for 1h. Cells were washed off the plates with LB and transferred into 50 ml LB supplemented with 12.5 μg/ml kanamycin and 10 μg/ml tetracycline. Cells were grown at 37°C for 4h, harvested and plated on LB agar plates supplemented with 12.5 μg/ml kanamycin, 10 μg/ml tetracycline, 2 % glucose and 2.5 mM 4-CP. Strains were single colony purified, and verified by marker analysis and colony PCR followed by XhoI digestion and Sanger sequencing. Strains with desired point mutations were cured of pKW20 by repeated growth in LB in absence of antibiotics, diluted 1:10 6 and plated on TYE plates. Strains were verified by marker analysis and Sanger sequencing of PCR products covering the targeting region. Strains used in Figure 5c were further whole genome sequenced on a MiSeq (Illumina). NGS data were analysed using breseq v0.35.1 78 .
Strains are listed in Supplementary Table T6. Annotated shuttle vectors and genomic loci are provided in Supplementary Data D1 and D3, respectively.

Assessment of growth and cell elongation phenotypes
Growth on solid media-Strains were streaked on the same TYE plate and incubated at 37°C overnight. The next morning, strains were re-streaked on a fresh TYE plate and incubated at 37°C for 12h.
Growth in liquid media-Strains were grown overnight in LB at 37°C. Cells were diluted 1/1000 into fresh LB into a 96-well flat bottom plate in octuplicate. The plate was incubated at 37°C in a Tecan microplate reader with regular shaking. Absorbance at 600 nm wavelength was measured every 5 min for 24h. OD 600 values were background corrected, normalised to the maximum OD 600 value of each well and averaged. Individual data points and the means were plotted.
DIC imaging of exponential phase cultures-Strains were grown overnight in LB at 37°C. The next day, cells were diluted 1/1000 into fresh LB and incubated at 37°C. 2-3 μl of exponential phase cells (OD 600 = 0.2-0.3) were applied onto an agarose pad and imaged on a Nikon Eclipse E800 microscope equipped with a 100x oil objective and a Photometrics Iris 9 CMOS camera using a differential interference contrast (DIC) imaging setup. Presented images were contrast-adjusted for display purposes.

Extended Data
Extended Data Figure 1. The position of the IC domain within the FtsA monomer varies.  linking is highlighted on the VmFtsA double filament structure (PDB 7Q6F). C β -C β distances between intermolecular P98C mutations are indicated by dotted lines. The inset provides a comparison between experimentally sampled intermolecular C β -C β distances by single cysteine point mutations (orange) and all intermolecular C β -C β distances between amino acids P98, S118, E199 and S252 (blue). Single cysteine point mutations sample intermolecular distances similar to those between double cysteine mutations. Calculated intermolecular C β -C β distances were rounded to one digit and duplicate values removed prior to plotting.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts a, Logical steps towards a divisome primed for curvature-guided septal PG synthesis. The temporal order of events remains to be determined. FtsZ filaments recruited to midcell are unaligned (left). Downstream divisome proteins are recruited and condensed into the narrow midcell plane by treadmilling FtsZ filaments that themselves will also be aligned through their interaction with curvature-sensing FtsA filaments (middle). Condensed complexes are aligned with the short axis of the cell by FtsA double filaments because of their curvature-sensing mechanism, which we propose here they share with MreB. Finally, FtsZ filaments distribute divisome components via treadmilling and might thereby reinforce their alignment with the short axis of the cell. FtsN is required for FtsA double filament formation and activation of septal PG synthases. By coordinating both activities across the inner membrane, FtsN might constitute a synchronising activation switch for the divisome, which allows circumferential synthesis of septal peptidoglycan through the alignment activity, and at the same time cell constriction to commence through the activation of PG synthesis (right). b, Schematic overview of core components of the divisome and elongasome highlighting the evolutionary relationship between the two complexes. We propose here that both complexes utilise the curvature-sensing properties of their cytoplasmic actin double filament scaffolds (red) to direct peptidoglycan synthesis around the cell's circumference. The bipartite PG synthases (FtsWI and RodA-PBP2, purple) are connected to the actin double filaments via integral membrane proteins that serve as structural and regulatory subunits (yellow), or potentially even directly. Unlike the elongasome, the divisome, in FtsN, possesses an additional regulatory subunit that might be necessary since cell division is regulated during the cell cycle. FtsZ is absent in the elongasome. FtsZ localises the divisome and its activities to midcell and probably more importantly into a narrow plane, an activity that is neither required nor desired in the elongasome.